This invention relates to a member of the fibroblast growth factor family designated FGF-9 and, more particularly, to an animal model for studying the role of FGF-9 in the regulation of cardiovascular and pulmonary physiology and growth. FNT (Note: Literature references on the following background information and on conventional test methods and laboratory procedures well-known to the person skilled in the art, and other such state-of-the-art techniques as used herein, are indicated by reference numbers in brackets, and appended at the end of the specification).
The first fibroblast growth factor (FGF) discovered in the 1970s, now known as FGF-2, had an activity that stimulated the proliferation of 3T3 fibroblasts. The FGF family has subsequently grown to include fifteen structurally related proteins, namely FGFs 1-10, FHFs 1-4 (fibroblast growth factor homologous factors) [1,2] and FGF-15. FGFs 1-10 interact with four distinct high-affinity FGF receptors (FGFRs) with varying affinity and specificity [3].
Alternative mRNA splicing significantly modifies the binding specificities of FGF receptors 1-3 [4]. An additional mechanism regulating FGF activity involves heparin or heparan sulfate proteoglycans (HSPG), molecules which are required for ligand-receptor interactions in vitro and possibly in vivo.
By sequence analysis, FHFs 1-4 show significant amino acid homology with other members of the FGF family [1]. However, thus far FHFs do not have any biological activity towards the four known FGF receptors.
Both FGF ligands and receptors are expressed in specific spatial and temporal patterns during embryonic development. Different FGFs and FGFRs display both overlapping and unique patterns of expression raising the question of specific function and redundancy of function. These issues can be addressed by examining the phenotypic consequences of mutations in FGF ligands and receptors in both human and experimental animal models. As shown by Santos-Ocampo et al., J. Biol. Chem. 271, 1726-1731 (1996), receptor specificity is an essential mechanism of FGFs.
Several human genetic diseases result from dominant gain of function mutations in the genes encoding FGFR 1, 2 and 3. These disorders result in skeletal dysplasias and/or craniosynostosis and some of these diseases have associated distinct CNS abnormalities.
The genetic disorders involving FGF receptor 3 (FGFR3) (achondroplasia, thanatophoric dysplasia, hypochondroplasia) demonstrate that FGFR3 controls the rate of normal growth of the skeleton [5] and may be an important signaling molecule in the developing temporal lobe and hippocampus [6].
The genetic disease, Apert's syndrome, which results from a point mutation in the FGF receptor 2 gene, is also associated with skeletal, cardiovascular, genitourinary and CNS phenotypes [7].
The biologic response to an FGF ligand depends on the cell-type, tissue and developmental stage. FGF is required for survival and growth of endothelial cells [8], 3T3 cells [9], receptor-expressing lymphoid cells [10], neurons and muscle.
FGF-2 is also effective in maintaining certain hematopoietic lineages in long term primary bone marrow culture [11] and for the survival and possible differentiation of hematopoietic progenitor cells [12].
During embryonic development FGFs are thought to be important for the induction and patterning of mesoderm [13-15]. The diverse types of responses to FGF (cell growth, survival and differentiation) may result from the activity of alternative signaling pathways in different cell-types, or from committed cells responding differentially to a common signaling pathway.
In the cardiovascular system, both FGF-1 and FGF-2 are proven mitogens for vascular endothelial cells and vascular smooth muscle cells.
For example, following balloon catheter endarterectomy, endothelial cell outgrowth into the denuded area is significantly enhanced by intravenously administered FGF-2 [16]. Following vascular surgery of trauma, reendothelialization is often incomplete. This may contribute to prolonged vascular disease and the failure of some surgical procedures (reviewed in [17]). The ability of FGF-2 to enhance endothelial outgrowth may therefore be a useful therapeutic tool [17]. PA1 Similarly, after prolonged infusion, FGF-2 significantly enhances intimal thickening [17]. PA1 FGF-1 also causes intimal thickening when over-expressed in arteries in vivo [18].
Endotheial and vascular smooth muscle cell growth is complex and probably involves a balance between both positive and negative regulatory molecules. Although FGF-2 is present in the arterial wall and is produced by both endothelial cells and vascular smooth muscle, it has little effect on the growth of normal vascular endothelial cells [19].
Nevertheless, FGF can stimulate the growth of injured endothelium. Both FGF-1 and FGF-2 lack signal peptides and are released from cells by poorly defined mechanisms. One hypothesis that accounts for these features of FGF-1 and FGF-2 proposes that cell injury, cell migration and cell death may result in the release into the extracellular environment of FGF, which would mediate a physiological response [17,20].
Unlike FGFs 1 and 2, many of the other FGFs are efficiently secreted. The specific spatial and temporal patterns of expression of these FGFs along with a growing body of genetic evidence (mutant mice and knockout mice) suggest that these FGFs are essential regulators of embryonic development.
The precise role of FGF signalling in cardiovascular development is not known. However, the expression of both FGF ligands and receptors in both cardiac and vascular mesoderm and endoderm suggest an important role for these molecules in development.
Additionally, FGFs 1, 2 and 4 can support the proliferation and differentiation of chick precardiac myoblasts. This activity can be blocked by chlorate, a known inhibitor of FGF ligand-receptor interactions [21,22]. The knockout of FGF-2 in mice, surprisingly, has little or no developmental consequences. Mice lacking either FGF 4 or 8 die too early in embryonic development to allow analysis of the role of these factors in cardiovascular development. The knockout of FGF-3 causes early inner ear and axial skeletal phenotypes, and the knockout of FGF 5 or 7 results in abnormal hair growth [23-25].
The four FGF receptor genes have also been disrupted in mice. A null mutation in the FGFR1 gene results in early embryonic lethality. These mice die around the time of gastrulation. Analysis of these animals demonstrates possible patterning defects in axial mesoderm as well as general growth retardation [14,15].
These studies suggest that FGFR1 is essential for normal rates of mesodermal cell proliferation and migration. FGFR1 is expressed in a wide variety of tissues later in development, however the embryonic lethality of FGFR1 null mice precludes a determination of the role of FGFR1 at these times.
FGFR3 null mice demonstrate defects in skeletal growth and in inner ear development [26]. The skeletal phenotype suggests that FGFR3 is a negative regulator of endochondral ossification and the inner ear phenotype demonstrates that FGFR3 is essential for the normal development of supporting cells within the organ of Corti. FGFR3 is expressed in many other tissues. However, a phenotype in these tissues is not readily apparent. Therefore, the function of FGFR3 in these tissues may be subtle or redundant with other FGFRs.
FGFR2 and FGFR4 have also been disrupted in mice. FGFR2 null mice die early in embryogenesis and FGFR4 null mice have little or no apparent phenotype.